- Top of page
- Materials and methods
- Supporting Information
Erythropoietin (Epo) is the major regulator of differentiation, proliferation and survival of erythroid progenitors, but the Epo-induced changes in gene expression that lead to these effects are not fully understood. The aim of this study was to examine how Epo, via activation of phosphatidylinositol 3-kinase (PI3K)/Akt, exerts its role in the development of erythroid progenitors from CD34+ cells, and to identify early Epo target genes in human erythroid progenitors. In CD34+ progenitor cells, Epo alone was able to induce cell cycle progression as demonstrated by upregulation of cyclin D3, E and A leading to hyperphosphorylation of the retinoblastoma protein (RB). These effects were completely counteracted by the PI3K inhibitor LY294002. Furthermore, enforced expression of an activated form of Akt kinase highly augmented Epo-induced erythropoiesis. Fluorescent-activated cell sorting (FACS)-sorted CD34+CD71+CD45RA−GPA− erythroid progenitors stimulated with Epo in the presence or absence of LY294002 were subjected to gene expression profiling. Several novel target genes of Epo were identified, and the majority were regulated in a PI3K-dependent manner, including KIT (CD117) and CDH1 (E-cadherin). FACS analysis of Epo-stimulated erythroid progenitors showed that the increased mRNA expression of KIT and CDH1 was accompanied by an induction of the corresponding proteins CD117 and E-cadherin.
Human erythropoiesis takes place in the bone marrow, where multipotent stem cells differentiate into mature erythrocytes. The earliest committed cells of the erythroid lineage, the erythroid burst-forming unit (BFU-E), express the erythropoietin receptor (EpoR), and progression through further developmental stages are completely dependent on signalling through this receptor (Krantz, 1991). Epo alone is able to induce erythroid development, but this process is enhanced by other cytokines, of which the most prominent is stem cell factor (Scf) (McNiece et al, 1991).
Activation of the signalling pathways upon ligand binding to EpoR is dependent on conformational changes of the receptor that bring together the dimerised receptor and Janus kinase 2 (JAK2) (Remy et al, 1999). Autophosphorylation of JAK2, followed by phosphorylation of cytoplasmic tyrosine residues of the EpoR results in an activated ligand-receptor complex that recruits several signalling molecules and adapter proteins that act as substrates for JAK2. Thus, several signalling cascades are involved in mediating the Epo-induced changes in gene expression and subsequent effects on differentiation, proliferation and survival (Richmond et al, 2005). Signal transducer and activator of transcription (STAT)5a/b, but also STAT1 and STAT3, have been shown to play a role in signal transduction, and JAK2 phosphorylation leads to dimerisation of these transcription factors and subsequent gene activation (Wojchowski et al, 1999; Richmond et al, 2005). The Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway is activated upon recruitment of the Grb2-Sos adapter molecules to the EpoR and this pathway also participates in mitogenesis (Jacobs-Helber & Sawyer, 2004; Arcasoy & Jiang, 2005).
The phosphatidylinositol 3-kinase (PI3K)-signalling cascade is crucial in mediating signals for survival and proliferation and is necessary for maturation of erythroid progenitors (Myklebust et al, 2002; Bouscary et al, 2003). PI3K can be activated by direct binding of the p85 regulatory subunit to the activated EpoR, and indirectly through binding to adapter molecules. The lipid products of PI3K activate a plethora of targets, including the protein kinase B/Akt. The PI3K-Akt pathway has been shown to play a central role in regulation of apoptosis and proliferation in several systems, including normal erythroid progenitors (Haseyama et al, 1999; Uddin et al, 2000). In the context of Epo signalling it has been shown that Akt kinase, which is activated by PI(3,4,5)P3 or PI(3,4)P2, phosphorylates both GATA-1 and Foxo3a, transcription factors of crucial importance in erythropoiesis (Uddin et al, 2000; Bouscary et al, 2003; Kadri et al, 2005).
In the present study we have investigated how Epo, by activating PI3K, exerts its effects on cell cycle progression and differentiation in human CD34+ progenitor cells. By use of genome wide expression profiling of Epo-stimulated CD34+CD71+CD45RA−GPA− erythroid progenitors, we identified novel Epo target genes and confirmed their differential expression by fluorescent-activated cell sorting (FACS) analysis, as well as hypothesise novel roles for signalling pathways that have no previously established function in erythropoiesis.
- Top of page
- Materials and methods
- Supporting Information
Erythropoietin provides the essential signals for differentiation and survival to erythroid progenitors. We investigated the role of PI3K-dependent Epo signalling in cell cycle progression of CD34+ progenitors, and showed that Epo alone was able to induce hyperphosphorylation of RB and upregulate cyclin D3, E and A. Genome wide profiling of CD34+CD71+CD45RA−GPA− erythroid progenitors showed that Epo initiated a transcriptional programme that led to significant change of expression levels in 584 genes after 4 h. The majority of these genes were apparently regulated in a PI3K-dependent manner, including glycophorin A, e-cadherin and Scf receptor/CD117 (KIT).
We have previously shown that Epo-induced differentiation of erythroid cells is dependent on PI3K/Akt signalling pathway (Myklebust et al, 2002). Here we showed that enforced expression of Myr-Akt, a constitutively active form of Akt, highly augmented the Epo-induced differentiation. Furthermore, Myr-Akt transfected cells were hypersensitive to Epo. Interestingly, Ghaffari and co-workers recently demonstrated that enforced expression of activated Akt in murine fetal liver progenitor cells overrode the need for Epo to induce erythroid differentiation (Ghaffari et al, 2006). They concluded that the enhanced erythroid maturation of activated-Akt-transduced cells was not limited to its anti-apoptotic or proliferative effect as no increase in total cell number was observed. Increased activation of the PI3K/Akt signalling pathway may be particularly relevant to patients with polycythemia vera (PV). PV is a clonal haematopoietic progenitor cell disease characterised by enhanced erythropoiesis and increased number of erythrocytes. PV erythroid progenitors are hypersensitive to several cytokines, including Epo (Casadevall et al, 1982). Upon stimulation with Epo or Scf, PV erythroid progenitors showed a marked increase in phosphorylation of Akt and its downstream targets GSK3α/β, compared with normal erythroid progenitors (Dai et al, 2005). Furthermore, the cytokine hypersensitivity and increased phosphorylation of Akt is probably caused by an activating mutation in JAK2 found in the majority of PV patients (Campbell et al, 2005; James et al, 2005; Zhao et al, 2005).
The molecular events that initiate cell cycle progression and differentiation upon Epo signalling in CD34+ progenitors are not fully understood. Although, several different pathways are activated, the critical role of PI3K has been shown (Myklebust et al, 2002; Bouscary et al, 2003; Schmidt et al, 2004). Here we show that Epo induced upregulation of cyclin D3, E and A in a PI3K-dependent manner, whereas the protein expression of p27Kip1 and p21Cip1 largely was unaffected. Bouscary et al (2003) also found a PI3K-dependent upregulation of D cyclins upon Epo stimulation and they also observed downregulation of p27Kip1, which was mediated by degradation by the E3 ligase SCFSKP2 (Bouscary et al, 2003). However, in our study, the erythroid progenitors represent an earlier developmental stage (BFU-E). It is possible that Epo-induced effects on cell cycle regulators differ in early versus later stages of erythroid development, as late erythroid precursors in response to Epo exit cell cycle during differentiation. Moreover, the regulation of cyclins is most likely not regulated at the transcription level, as none of the cyclins or CKIs were among the significantly changed genes by gene expression profiling. Furthermore, none of the cyclins that we tested in real time RT-PCR experiments showed significant changes in response to Epo stimulation.
Genome wide profiling has been used in several studies of erythroid differentiation (Kolbus et al, 2003; Edvardsson et al, 2004; Fujishima et al, 2004; Welch et al, 2004). The findings of these studies are heterogeneous, reflecting the variation in the experimental systems used. By choosing freshly isolated early erythroid progenitors (CD34+CD71+CD45RA−GPA−), and by stimulating them for 4 h with Epo with or without LY294002, our study design aimed to discover early targets of Epo signalling in the human system and to clarify whether their regulation was PI3K-dependent. By choosing a threshold of 1·5-fold regulation and applying a practical two-step filtering procedure, we defined a list of 584 unique known genes that exhibited regulation upon Epo stimulation. This approach has been shown to be practical in defining a list of genes that contains physiologically relevant targets (Shen et al, 2004). A number of known Epo target genes were included in our list of upregulated genes. GYPA and TFRC are associated with erythroid differentiation and were upregulated in a PI3K-dependent fashion. This was shown in the microarray experiments and verified at the protein level by FACS assays. One of the most interesting findings in our study was the upregulation of KIT (CD117) expression in response to Epo treatment. Scf acts synergistically with Epo to enhance the generation of glycophorin A positive cells from CD34+ progenitors (Wu et al, 1997; Myklebust et al, 2002; Munugalavadla et al, 2005). Several mechanisms of cooperative action between Epo and Scf have been suggested, and it is well established that Scf can promote an increase in EpoR expression (Lodish et al, 1995; Kapur & Zhang, 2001). Upregulation of KIT (CD117) in response to Epo is a novel mechanism for such cooperative action. Our data also clearly indicated that the Epo-induced KIT upregulation is dependent on activation of PI3K. The PI3K inhibitor LY294002 abrogated the increased KIT expression, and in the cultures that were treated with LY294002 only, the levels of KIT mRNA and protein were even lower compared with the medium only treated cultures. This suggests that, for CD34+ progenitors, a certain basal level of PI3K activity is required to maintain expression of KIT. KIT is downregulated (repressed by GATA-1) in the later stages of erythroid differentiation, but the levels in CD34+ progenitors are high (Welch et al, 2004; Munugalavadla et al, 2005). In our experimental system, Scf could not bypass the requirement for Epo and alone promote erythroid differentiation of CD34+ progenitors (data not shown). Previous data on c-kit dependency of early erythroid progenitors are controversial. Human BFU-Es have been shown to be Scf-dependent when cultured in serum free media (Dai et al, 1991), but it has also been shown that c-Kit w/w mice, that are functionally c-kit deficient, can be rescued by transgenic, enforced expression of Epo (Waskow et al, 2004).
CDH1 was, in addition to KIT, among the intriguing candidate genes from the microarray screening that we decided to analyse at the protein level. This gene encodes for a cell-cell adhesion glycoprotein that is expressed at the erythroblast and the normoblast stages, and it has previously been shown that e-cadherin has a functional role in erythropoiesis (Armeanu et al, 2000). We have now shown that e-cadherin is rapidly induced by Epo in early CD34+ progenitors. The protein expression was restricted to the CD71high expressing cells. By use of the PubGene tool we identified literature neighbourhoods that consisted of Epo-regulated genes defined by our SAM. The neighbourhood of IL6ST, which obtained the highest score, included both known Epo targets, such as transferrin receptor, haemoglobin alpha 2 and glycophorin A, and also KIT that was validated in our study. Upregulation of IL1R1 and EGFR, could provide mechanisms through which survival and proliferation are promoted, in response to interleukin 1 or epidermal growth factor stimulation respectively. Interestingly, PTPRC (CD45), was also among the upregulated genes in this cluster. Several studies have shown that CD45 plays an important role in the negative regulation of erythroid differentiation (Harashima et al, 2002). Thus, the PubGene tool can be helpful in organising the differentially expressed genes into functionally related gene clusters, and aids the generation of novel hypotheses.
Taken together, our data show that ligand binding to the Epo receptor in early CD34+ progenitors initiates a plethora of signalling cascades. The physiological effects of Epo on survival, proliferation and differentiation at this stage are completely dependent on functional PI3K, and the observed expression changes of important target genes reflect this dependency.